The long-term goal of this proposal is to elucidate the molecular interactions involved in the sorting, transport, and integration of membrane components into various regions of myelin internode. Studies will focus on the sorting and transport of oligodendrocyte and myelin proteins and will investigate both exocytic and endocytic pathways. Specific Aim 1 will determine the ultrastructural localization of myelin proteins and characterize the role that microtubules play in their transport. These studies will extend previous work that has described where myelin protein are synthesized and where they are localized within the myelin sheath. Whether microtubule depolarization alters the morphology of myelinating oligodendrocytes and the intracellular distribution of myelin proteins will be determined. If microtubules are involved in myelin protein transport, myelin proteins will accumulate in oligodendrocyte perinuclear cytoplasm. Other studies will investigate basic features of oligodendrocyte microtubules, including their orientation (polarity), their molecular composition, their association with cytoplasmic organelles, and their 3- dimensional relationship with the Golgi apparatus and microtubule organizing center. These studies will employ state of the art technologies including EM immunocytochemistry, confocal microscopy, and in situ hybridization. In addition to the delivery of proteins to surface membranes, oligodendrocytes retrieve and redistribute surface membrane components during active myelination. Specific Aim 2 will characterize receptor- mediated endocytosis in myelinating oligodendrocytes. Electron microscopic studies will determine where this process occurs and whether it is developmentally regulated by determining the subcellular distribution of clathrin-coated pits. Other studies will characterize intracellular trafficking of endocytosed molecules by double-labelling EM immunocytochemistry. The third specific aim will investigate myelin protein-sorting in Quaking mice. Dysmyelination in Quaking is thought to include alterations in the processing and transport in myelin components. The proposed studies will represent the first systematic investigation of protein-sorting and receptor-mediated endocytosis in oligodendrocytes and they should provide fundamental understanding of basic aspects of these processes as they relate to myelinogenesis and dysmyelinating diseases.